Protein O-Fucosyltransferase 1 Expression Impacts Myogenic C2C12 Cell Commitment via the Notch Signaling Pathway

The Notch signaling pathway plays a crucial role in skeletal muscle regeneration in mammals by controlling the transition of satellite cells from quiescence to an activated state, their proliferation, and their commitment toward myotubes or self-renewal. O-fucosylation on Notch receptor epidermal growth factor (EGF)-like repeats is catalyzed by the protein O-fucosyltransferase 1 (Pofut1) and primarily controls Notch interaction with its ligands. To approach the role of O-fucosylation in myogenesis, we analyzed a murine myoblastic C2C12 cell line downregulated for Pofut1 expression by short hairpin RNA (shRNA) inhibition during the time course of differentiation. Knockdown of Pofut1 affected the signaling pathway activation by a reduction of the amount of cleaved Notch intracellular domain and a decrease in downstream Notch target gene expression. Depletion in Pax7⁺/MyoD⁻ cells and earlier myogenic program entrance were observed, leading to an increase in myotube quantity with a small number of nuclei, reflecting fusion defects. The rescue of Pofut1 expression in knockdown cells restored Notch signaling activation and a normal course in C2C12 differentiation. Our results establish the critical role of Pofut1 on Notch pathway activation during myogenic differentiation.     

Divergent and conserved roles of Dll1 signaling in development of craniofacial and trunk muscle

Craniofacial and trunk skeletal muscles are evolutionarily distinct and derive from cranial and somitic mesoderm, respectively. Different regulatory hierarchies act upstream of myogenic regulatory factors in cranial and somitic mesoderm, but the same core regulatory network – MyoD, Myf5 and Mrf4 – executes the myogenic differentiation program. Notch signaling controls self-renewal of myogenic progenitors as well as satellite cell homing during formation of trunk muscle, but its role in craniofacial muscles has been little investigated. We show here that the pool of myogenic progenitor cells in craniofacial muscle of Dll1^LacZ/Ki mutant mice is depleted in early fetal development, which is accompanied by a major deficit in muscle growth. At the expense of progenitor cells, supernumerary differentiating myoblasts appear transiently and these express MyoD. The progenitor pool in craniofacial muscle of Dll1^LacZ/Ki mutants is largely rescued by an additional mutation of MyoD. We conclude from this that Notch exerts its decisive role in craniofacial myogenesis by repression of MyoD. This function is similar to the one previously observed in trunk myogenesis, and is thus conserved in cranial and trunk muscle. However, in cranial mesoderm-derived progenitors, Notch signaling is not required for Pax7 expression and impinges little on the homing of satellite cells. Thus, Dll1 functions in satellite cell homing and Pax7 expression diverge in cranial- and somite-derived muscle.     

Constitutive Notch activation upregulates Pax7 and promotes the self-renewal of skeletal muscle satellite cells

Notch signaling is a conserved cell fate regulator during development and postnatal tissue regeneration. Using skeletal muscle satellite cells as a model and through myogenic cell lineage-specific NICD(OE) (overexpression of constitutively activated Notch 1 intracellular domain), here we investigate how Notch signaling regulates the cell fate choice of muscle stem cells. We show that in addition to inhibiting MyoD and myogenic differentiation, NICD(OE) upregulates Pax7 and promotes the self-renewal of satellite cell-derived primary myoblasts in culture. Using MyoD(-/-) myoblasts, we further show that NICD(OE) upregulates Pax7 independently of MyoD inhibition. In striking contrast to previous observations, NICD(OE) also inhibits S-phase entry and Ki67 expression and thus reduces the proliferation of primary myoblasts. Overexpression of canonical Notch target genes mimics the inhibitory effects of NICD(OE) on MyoD and Ki67 but not the stimulatory effect on Pax7. Instead, NICD regulates Pax7 through interaction with RBP-Jκ, which binds to two consensus sites upstream of the Pax7 gene. Importantly, satellite cell-specific NICD(OE) results in impaired regeneration of skeletal muscles along with increased Pax7(+) mononuclear cells. Our results establish a role of Notch signaling in actively promoting the self-renewal of muscle stem cells through direct regulation of Pax7.     

Notch signaling deficiency underlies age-dependent depletion of satellite cells in muscular dystrophy

Duchenne muscular dystrophy (DMD) is a devastating disease characterized by muscle wasting, loss of mobility and death in early adulthood. Satellite cells are muscle-resident stem cells responsible for the repair and regeneration of damaged muscles. One pathological feature of DMD is the progressive depletion of satellite cells, leading to the failure of muscle repair. Here, we attempted to explore the molecular mechanisms underlying satellite cell ablation in the dystrophin mutant mdx mouse, a well-established model for DMD. Initial muscle degeneration activates satellite cells, resulting in increased satellite cell number in young mdx mice. This is followed by rapid loss of satellite cells with age due to the reduced self-renewal ability of mdx satellite cells. In addition, satellite cell composition is altered even in young mdx mice, with significant reductions in the abundance of non-committed (Pax7⁺ and Myf5⁻) satellite cells. Using a Notch-reporter mouse, we found that the mdx satellite cells have reduced activation of Notch signaling, which has been shown to be necessary to maintain satellite cell quiescence and self-renewal. Concomitantly, the expression of Notch1, Notch3, Jag1, Hey1 and HeyL are reduced in the mdx primary myoblast. Finally, we established a mouse model to constitutively activate Notch signaling in satellite cells, and show that Notch activation is sufficient to rescue the self-renewal deficiencies of mdx satellite cells. These results demonstrate that Notch signaling is essential for maintaining the satellite cell pool and that its deficiency leads to depletion of satellite cells in DMD.KEY WORDS: Muscular dystrophy, Notch signaling, Stem cell     

Syndecan-3 and Notch cooperate in regulating adult myogenesis

Skeletal muscle postnatal growth and repair depend on satellite cells and are regulated by molecular signals within the satellite cell niche. We investigated the molecular and cellular events that lead to altered myogenesis upon genetic ablation of Syndecan-3, a component of the satellite cell niche. In the absence of Syndecan-3, satellite cells stall in S phase, leading to reduced proliferation, increased cell death, delayed onset of differentiation, and markedly reduced numbers of Pax7⁺ satellite cells accompanied by myofiber hypertrophy and an increased number of centrally nucleated myofibers. We show that the aberrant cell cycle and impaired self-renewal of explanted Syndecan-3–null satellite cells are rescued by ectopic expression of the constitutively active Notch intracellular domain. Furthermore, we show that Syndecan-3 interacts with Notch and is required for Notch processing by ADAM17/tumor necrosis factor-α–converting enzyme (TACE) and signal transduction. Together, our data support the conclusion that Syndecan-3 and Notch cooperate in regulating homeostasis of the satellite cell population and myofiber size.     

The thymus regulates skeletal muscle regeneration by directly promoting satellite cell expansion

The thymus is the central immune organ, but it is known to progressively degenerate with age. As thymus degeneration is paralleled by the wasting of aging skeletal muscle, we speculated that the thymus may play a role in muscle wasting. Here, using thymectomized mice, we show that the thymus is necessary for skeletal muscle regeneration, a process tightly associated with muscle aging. Compared to control mice, the thymectomized mice displayed comparable growth of muscle mass, but decreased muscle regeneration in response to injury, as evidenced by small and sparse regenerative myofibers along with inhibited expression of regeneration-associated genes myh3, myod, and myogenin. Using paired box 7 (Pax7)-immunofluorescence staining and 5-Bromo-2′-deoxyuridine-incorporation assay, we determined that the decreased regeneration capacity was caused by a limited satellite cell pool. Interestingly, the conditioned culture medium of isolated thymocytes had a potent capacity to directly stimulate satellite cell expansion in vitro. These expanded cells were enriched in subpopulations of quiescent satellite cells (Pax7^highMyoD^lowEdU^pos) and activated satellite cells (Pax7^highMyoD^highEdU^pos), which were efficiently incorporated into the regenerative myofibers. We thus propose that the thymus plays an essential role in muscle regeneration by directly promoting satellite cell expansion and may function profoundly in the muscle aging process.     

Brain-derived Neurotrophic Factor Regulates Satellite Cell Differentiation and Skeltal Muscle Regeneration

In adult skeletal muscle, brain-derived neurotrophic factor (BDNF) is expressed in myogenic progenitors known as satellite cells. To functionally address the role of BDNF in muscle satellite cells and regeneration in vivo, we generated a mouse in which BDNF is specifically depleted from skeletal muscle cells. For comparative purposes, and to determine the specific role of muscle-derived BDNF, we also examined muscles of the complete BDNF^−/− mouse. In both models, expression of the satellite cell marker Pax7 was significantly decreased. Furthermore, proliferation and differentiation of primary myoblasts was abnormal, exhibiting delayed induction of several markers of differentiation as well as decreased myotube size. Treatment with exogenous BDNF protein was sufficient to rescue normal gene expression and myotube size. Because satellite cells are responsible for postnatal growth and repair of skeletal muscle, we next examined whether regenerative capacity was compromised. After injury, BDNF-depleted muscle showed delayed expression of several molecular markers of regeneration, as well as delayed appearance of newly regenerated fibers. Recovery of wild-type BDNF levels was sufficient to restore normal regeneration. Together, these findings suggest that BDNF plays an important role in regulating satellite cell function and regeneration in vivo, particularly during early stages.     

Skeletal Muscle Satellite Cells Are Committed to Myogenesis and Do Not Spontaneously Adopt Nonmyogenic Fates

The developmental potential of skeletal muscle stem cells (satellite cells) remains controversial. The authors investigated satellite cell developmental potential in single fiber and clonal cultures derived from MyoD^iCre/+;R26R^EYFP/+ muscle, in which essentially all satellite cells are permanently labeled. Approximately 60% of the clones derived from cells that co-purified with muscle fibers spontaneously underwent adipogenic differentiation. These adipocytes stained with Oil-Red-O and expressed the terminal differentiation markers, adipsin and fatty acid binding protein 4, but did not express EYFP and were therefore not of satellite cell origin. Satellite cells mutant for either MyoD or Myf-5 also maintained myogenic programming in culture and did not adopt an adipogenic fate. Incorporation of additional wash steps prior to muscle fiber plating virtually eliminated the non-myogenic cells but did not reduce the number of adherent Pax7+ satellite cells. More than half of the adipocytes observed in cultures from Tie2-Cre mice were recombined, further demonstrating a non-satellite cell origin. Under adipogenesis-inducing conditions, satellite cells accumulated cytoplasmic lipid but maintained myogenic protein expression and did not fully execute the adipogenic differentiation program, distinguishing them from adipocytes observed in muscle fiber cultures. The authors conclude that skeletal muscle satellite cells are committed to myogenesis and do not spontaneously adopt an adipogenic fate.     

Increased Corneal Epithelial Turnover Contributes to Abnormal Homeostasis in the Pax6+/? Mouse Model of Aniridia

We aimed to test previous predictions that limbal epithelial stem cells (LESCs) are quantitatively deficient or qualitatively defective in Pax6^+/− mice and decline with age in wild-type (WT) mice. Consistent with previous studies, corneal epithelial stripe patterns coarsened with age in WT mosaics. Mosaic patterns were also coarser in Pax6^+/− mosaics than WT at 15 weeks but not at 3 weeks, which excludes a developmental explanation and strengthens the prediction that Pax6^+/− mice have a LESC-deficiency. To investigate how Pax6 genotype and age affected corneal homeostasis, we compared corneal epithelial cell turnover and label-retaining cells (LRCs; putative LESCs) in Pax6^+/− and WT mice at 15 and 30 weeks. Limbal BrdU-LRC numbers were not reduced in the older WT mice, so this analysis failed to support the predicted age-related decline in slow-cycling LESC numbers in WT corneas. Similarly, limbal BrdU-LRC numbers were not reduced in Pax6^+/− heterozygotes but BrdU-LRCs were also present in Pax6^+/− corneas. It seems likely that Pax6^+/− LRCs are not exclusively stem cells and some may be terminally differentiated CD31-positive blood vessel cells, which invade the Pax6^+/− cornea. It was not, therefore, possible to use this approach to test the prediction that Pax6^+/− corneas had fewer LESCs than WT. However, short-term BrdU labelling showed that basal to suprabasal movement (leading to cell loss) occurred more rapidly in Pax6^+/− than WT mice. This implies that epithelial cell loss is higher in Pax6^+/− mice. If increased corneal epithelial cell loss exceeds the cell production capacity it could cause corneal homeostasis to become unstable, resulting in progressive corneal deterioration. Although it remains unclear whether Pax6^+/− mice have LESC-deficiency, we suggest that features of corneal deterioration, that are often taken as evidence of LESC-deficiency, might occur in the absence of stem cell deficiency if corneal homeostasis is destabilised by excessive cell loss.     

Hypoxia promotes satellite cell self-renewal and enhances the efficiency of myoblast transplantation

Microenvironmental oxygen (O(2)) regulates stem cell activity, and a hypoxic niche with low oxygen levels has been reported in multiple stem cell types. Satellite cells are muscle-resident stem cells that maintain the homeostasis and mediate the regeneration of skeletal muscles. We demonstrate here that hypoxic culture conditions favor the quiescence of satellite cell-derived primary myoblasts by upregulating Pax7, a key regulator of satellite cell self-renewal, and downregulating MyoD and myogenin. During myoblast division, hypoxia promotes asymmetric self-renewal divisions and inhibits asymmetric differentiation divisions without affecting the overall rate of proliferation. Mechanistic studies reveal that hypoxia activates the Notch signaling pathway, which subsequently represses the expression of miR-1 and miR-206 through canonical Hes/Hey proteins, leading to increased levels of Pax7. More importantly, hypoxia conditioning enhances the efficiency of myoblast transplantation and the self-renewal of implanted cells. Given the robust effects of hypoxia on maintaining the quiescence and promoting the self-renewal of cultured myoblasts, we predict that oxygen levels in the satellite cell niche play a central role in precisely balancing quiescence versus activation, and self-renewal versus differentiation, in muscle stem cells in vivo.     

Silencing Pax3 by shRNA inhibits the proliferation and differentiation of duck (Anas platyrhynchos) myoblasts

The Pax3 gene has been proven to play a crucial role in determining myogenic progenitor cell fate during embryonic myogenesis; however, the molecular role of Pax3 in myoblast development during later stages of myogenesis is unknown. We hypothesized that Pax3 would function in myoblast proliferation and differentiation; therefore, we employed three short hairpin RNAs (shRNAs) (shRNA1, shRNA2, and shRNA3) that target Pax3 to characterize the function of Pax3 in duck myoblast development. The mRNA and protein expression levels of Pax3 in duck myoblasts were detected using real-time PCR and Western blotting. Cell proliferation was assessed using the MTT and BrdU assays, while cell differentiation was assayed using immunofluorescence labeling with a MyoG antibody. Additionally, folic acid (FA), which is a rescue tool, was added into the medium of duck myoblasts to indirectly examine the function of Pax3 on duck myoblast proliferation and differentiation. The results revealed that one of the shRNA vectors, shRNA1, could significantly and stably reduce the expression of Pax3 (P < 0.05). Silencing Pax3 by shRNA1 significantly reduced the proliferation and differentiation of duck myoblasts (P < 0.05) due to downregulated expression of myogenic regulator factors. These trends could be rescued by adding FA; and Pax7, a paralog gene of Pax3, was involved in those processes. Overall, Pax3 had a positive function in duck myoblast proliferation and differentiation by modulating the expression of myogenic regulation factors, and shRNA targeting of Pax3 might be a new approach for understanding the function of Pax3 in the development of diverse tissues.     

Notch1-mediated signaling regulates proliferation of porcine satellite cells (PSCs)

Notch signaling is an evolutionarily conserved cell–cell communication mechanism involved in the regulation of cell proliferation, differentiation and fate decisions of mammalian cells. In the present study, we investigated the possible requirement for Notch signaling in the proliferation and differentiation of porcine satellite cells. We show that Notch1, 2 and 3 are expressed in cultured porcine satellite cells. Knock-down of NOTCH1, but not NOTCH2 and NOTCH3, decreases the proliferation of porcine satellite cells. In contrast, enhancement of NOTCH1 expression via treatment of porcine satellite cells with recombinant NF-κB increases the proliferation of porcine satellite cells. The alteration of porcine satellite cell proliferation is associated with significant changes in the expression of cell cycle related genes (cyclin B1, D1, D2, E1 and p21), myogenic regulatory factors (MyoD and myogenin) and the Notch effector Hes5. In addition, alteration of Notch1 expression in porcine satellite cells causes changes in the expression of GSK3β-3. Taken together, these findings suggest that of the four notch-related genes, Notch1is likely to be required for regulating the proliferation and therefore the maintenance of porcine satellite cells in vivo, and do so through activation of the Notch effector gene Hes5.     

Type 2 cGMP-dependent protein kinase regulates homeostasis by blocking c-Jun N-terminal kinase in the colon epithelium

Analysis of knockout animals indicates that 3′,5′cyclic guanosine monophosphate (cGMP) has an important role in gut homeostasis but the signaling mechanism is not known. The goals of this study were to test whether increasing cGMP could affect colon homeostasis and determine the mechanism. We increased cGMP in the gut of Prkg2^+/+ and Prkg2^−/− mice by treating with the PDE5 inhibitor Vardenafil (IP). Proliferation, differentiation and apoptosis in the colon mucosa were then quantitated. Vardenafil (Vard) treatment increased cGMP in colon mucosa of all mice, but reduced proliferation and apoptosis, and increased differentiation only in Prkg2^+/+ mice. Vard and cGMP treatment also increased dual specificity protein phosphatase 10 (DUSP10) expression and reduced phospho-c-Jun N-terminal kinase (JNK) levels in the colon mucosa of Prkg2^+/+ but not Prkg2^−/− mice. Treatment of Prkg2^−/− mice with the JNK inhibitor SP600125 reversed the defective homeostasis observed in these animals. Activation of protein kinase G2 (PKG2) in goblet-like LS174T cells increased DUSP10 expression and reduced JNK activity. PKG2 also increased goblet cell-specific MUC2 expression in LS174T cells, and this process was blocked by DUSP10-specific siRNA. The ability of cGMP signaling to inhibit JNK-induced apoptosis in vivo was demonstrated using dextran sodium sulfate (DSS) to stress the colon epithelium. Vard was a potent inhibitor of DSS-induced epithelial apoptosis, and significantly blocked pathological endpoints in this model of experimental colitis. In conclusion, Vard treatment activates cGMP signaling in the colon epithelium. Increased PKG2 activity alters homeostasis by suppressing proliferation and apoptosis while promoting differentiation. The PKG2-dependent mechanism was shown to involve increased DUSP10 and subsequent inhibition of JNK activity.     

Regulation of Notch signaling and endocytosis by the Lgl neoplastic tumor suppressor

The evolutionarily conserved neoplastic tumor suppressor protein, Lethal (2) giant larvae (Lgl), plays roles in cell polarity and tissue growth via regulation of the Hippo pathway. In our recent study, we showed that in the developing Drosophila eye epithelium, depletion of Lgl leads to increased ligand-dependent Notch signaling. lgl mutant tissue also exhibits an accumulation of early endosomes, recycling endosomes, early-multivesicular body markers and acidic vesicles. We showed that elevated Notch signaling in lgl⁻ tissue can be rescued by feeding larvae the vesicle de-acidifying drug chloroquine, revealing that Lgl attenuates Notch signaling by limiting vesicle acidification. Strikingly, chloroquine also rescued the lgl⁻ overgrowth phenotype, suggesting that the Hippo pathway defects were also rescued. In this extraview, we provide additional data on the regulation of Notch signaling and endocytosis by Lgl, and discuss possible mechanisms by which Lgl depletion contributes to signaling pathway defects and tumorigenesis.